【摘要】 【目的】本研究旨在丰富西方蜜蜂Apis mellifera过氧化物还原酶5（peroxiredoxin-5） AmPRX5的理化性质和分子特征信息，检测AmPRX5的表达模式，并探究AmPRX5在蜜蜂球囊菌Ascosphaera apis侵染后对西方蜜蜂的抗氧化应激作用，为进一步开展AmPRX5功能和作用机制研究提供科学依据。【方法】通过PCR扩增西方蜜蜂AmPRX5的CDS;使用相关软件预测AmPRX5的理化性质和分子特征，构建蛋白互作网络，并进行保守基序、结构域和系统进化分析。采用RT-qPCR检测AmPRX5在西方蜜蜂工蜂不同发育阶段（卵、 3和5日龄幼虫、 1和2日龄预蛹、 1日龄蛹以及1, 2, 6, 8和12日龄成虫）和工蜂成虫不同组织（脑、咽下腺、中肠、触角、毒腺、表皮和脂肪体）及3日龄幼虫接种蜜蜂球囊菌后4, 5和6日龄幼虫肠道中的表达量；利用分光光度法检测蜜蜂球囊菌接种3日龄幼虫后4, 5和6日龄幼虫肠道中的过氧化氢（H₂O₂）含量。【结果】成功克隆到西方蜜蜂AmPRX5的CDS,长558 bp。AmPRX5的分子式为C₉₀₄H₁₄₄₅N₂₄₁O₂₆₄S₈,分子量约为20.17 kD,等电点为8.99,平均亲水系数为-0.013。AmPRX5含有19个磷酸化位点，但不含典型的信号肽和跨膜结构域。AmPRX5的二级结构包含88个无规卷曲、 59个α-螺旋和38条延伸链。AmPRX5与硫氧还蛋白还原酶等10个蛋白构成1个互作网络。西方蜜蜂和其他9种蜂的PRX5均包含1个相同的PRX5结构域和6个保守基序。西方蜜蜂与东方蜜蜂A.cerana的PRX5同源性最高。AmPRX5在1日龄蛹中的表达量最高且显著高于卵、 3日龄幼虫、 5日龄幼虫、 1日龄预蛹和2日龄预蛹中的表达量；AmPRX5在12日龄成虫体内的表达量最高且显著高于1, 2, 6和8日龄成虫体内的表达量；AmPRX5在成虫触角中的表达量最高且显著高于中肠、脂肪体、表皮、毒腺和脑中的表达量。与未接种对照组比较，西方蜜蜂3日龄幼虫接种蜜蜂球囊菌后AmPRX5的表达量在4日龄幼虫肠道中上调但无显著差异，在5日龄幼虫肠道中极显著上调，在6日龄幼虫肠道中显著上调；H₂O₂的含量在接种蜜蜂球囊菌后4和5日龄幼虫肠道中均极显著上升，在6日龄幼虫肠道中显著升高。【结论】AmPRX5是一种潜在的亲水性、非跨膜和胞内蛋白；AmPRX5在西方蜜蜂不同发育阶段和不同组织中动态差异表达；AmPRX5可能通过影响H₂O₂含量参与幼虫对蜜蜂球囊菌侵染的应答。更多还原

【Abstract】 【Aim】This study aims to enrich the information on the physical and chemical properties, and molecular characteristics of peroxiredoxin-5（AmPRX5） from the western honeybee, Apis mellifera, investigate the expression pattern of AmPRX5, and explore the role of AmPRX5 in antioxidant stress response in Apis mellifera following infection with Ascosphaera apis, so as to provide a scientific basis for further research on the function and action mechanism of AmPRX5. 【Methods】The CDS of AmPRX5 of Apis mellifera was amplified by PCR. Relevant software was used to predict the physicochemical properties and molecular characteristics of AmPRX5, construct the protein interaction network, identify conserved motifs and domains, and conduct phylogenetic analysis. RT-qPCR was employed to detect the expression levels of AmPRX5 in different developmental stages（egg, 3-and 5-day-old larvae, 1-and 2-day-old prepupa, 1-day-old pupa, and 1-, 2-, 6-, 8-and 12-day-old adults） of workers and different tissues（brain, hypopharyngeal gland, midgut, antenna, venom gland, cuticle and fat body） of worker adults and the guts of the 4-, 5-and 6-day-old larvae of Apis mellifera after inoculation of the 3-day-old larvae with Ascosphaera apis. The hydrogen peroxide（H₂O₂） contents in the guts of the 4-, 5-and 6-day-old larvae after inoculation of the 3-day-old larvae with Ascosphaera apis were detected by spectrophotometry. 【Results】The CDS of AmPRX5 of Apis mellifera was successfully cloned, with the length of 558 bp. AmPRX5 has the molecular formula of C₉₀₄H₁₄₄₅N₂₄₁O₂₆₄S₈, the molecular weight of approximately 20.17 kD, an isoelectric point of 8.99, and the average hydrophilic coefficient of-0.013. AmPRX5 contains 19 phosphorylation sites, and does not contain typical signal peptides and transmembrane domains. The secondary structure of AmPRX5 comprises 88 random coils, 59 α-helices and 38 extended chains. AmPRX5 forms an interaction network with 10 proteins, including thioredoxin reductase. PRX5 proteins from Apis mellifera and nine other bee species all contain an identical PRX5 domain and six conserved motifs. The PRX5 from Apis mellifera shares the highest homology with that from Apis cerana. The expression level of AmPRX5 in the 1-day-old pupae was the highest and significantly higher than those in the egg, 3-day-old larva, 5-day-old larva, 1-day-old prepupa and 2-day-old prepupa. The expression level of AmPRX5 in the 12-day-old adults was the highest and significantly higher than those in the 1-, 2-, 6-and 8-day-old adults. The expression level of AmPRX5 in the adult antenna was the highest and significantly higher than those in the midgut, fat body, cuticle, venom gland and brain. After inoculation of the 3-day-old larvae of Apis mellifera with Ascosphaera apis, the expression level of AmPRX5 in the gut of the 4-day-old larvae was up-regulated but showed no significant difference from that in the uninoculated control group, that in the gut of the 5-day-old larvae was extremely significantly up-regulated and that in the gut of the 6-day-old larvae was significantly up-regulated as compared with those in the uninoculated control group. After inoculation of the 3-day-old larvae with Ascosphaera apis, the H₂O₂ contents in the guts of the 4-and 5-day-old larvae increased extremely significantly and that in the gut of the 6-day-old larvae increased significantly as compared with those in the uninoculated control group. 【Conclusion】AmPRX5 is a potential hydrophilic, non-transmembrane and intracellular protein. AmPRX5 is expressed dynamically and differentially in different developmental stages and tissues of Apis mellifera. AmPRX5 may participate in the response of larvae to the infection of Ascosphaera apis by influencing the H₂O₂ content.更多还原